Shaped bodies of pullulan and their use

ABSTRACT

Pullulan, a water-soluble polysaccharide produced by Pullularia pullulans from conventional culture media and consisting of repeating units of maltotriose linked by Alpha -1,6 bonds, can be shaped by compression molding or extrusion at elevated temperature or by evaporation of water from its aqueous solutions to form shaped bodies, such as films or coatings which are practically impermeable to atmospheric oxygen in thin layers and unaffected by oils and fats so as to provide valuable packing materials for food, pharmaceuticals, and other oxygen sensitive materials. Pullulan is edible and biodegradable.

United States Patent Hljlya et al. [45] Ja 8, 1974 SHAPED BODIES 0FPULLULAN AND 3,406,114 10/1968 Goren 195/31 P THEIR USE [75] Inventors'gag";ggg g g iggs Primary Examiner-Theodore Morris Attorney-Hans Bermanand Kurt Kelman [73] Assignee: Hayashibara Biochemical Laboratories,Incorporated, Okayama-ken, Japan a [22 Filed: July 13, 1972 ABSTRACT[211 App]. No" 271,378 Pullulan, a water-soluble polysaccharide producedby 1 Pullularia pullulans from conventional culture media e gn Applicaion Priority ata and consisting of repeating .units of maltotrioselinked July 23, 1971 Japan 46/54579 y 01-16 ond can be shaped bycompression mold- Oct. 30, 1971 Japan 46/85960 ing of extrusion atelevated temperature of y p ration of water from its aqueous solutionsto form 52 us. c1. 106/139,, 106/213, 260/174 ST shaped bodies, such asfilms or coatings which are [51] Int. Cl. C08h l/00, C08h 7/00practically impermeable to atmospheric oxygen in thin [58] Field ofSearch 106/139, 162, 213; layer n un ffecte by oils and fats so as toprovide 1 260/174; 195/31 P valuable packing materials for food,pharmaceuticals, and other oxygen sensitive materials. Pullulan isedible [56] References Cited and biodegradable.

UNITED STATES PATENTS 3,054,689 9/1962 Jeans 106/208 14 Claims, N0Drawings 1 SHAPED BODIES OF PULLULAN AND THEIR USE This inventionrelates to pullulan, and particularly to shaped bodies of pullulan andto their use.

Pullulan is a polysaccharide consisting of maltotriose units linked bya-1,6bonds. As disclosed by H. Bender et al (Biochim. Biophys. Acta 36[1959] 309) and S.

' Ueda, (Kogyo Kagaku Zasshi 67 [1964] 757), pullulan is produced bystrains of Pullularia or Dematium growin g on conventional culturemedia, and is readily recovered from a culture medium because ofitsinsolubility in methanol. While the material has been known for arelatively long period, it did not find any practical applicationheretofore.

It has now been found that pullulan is readily converted into shapedbodies having valuable properties. The material is water soluble,edible, andbiodegradable. It can be converted to shaped bodies bydissolving it in water, imparting to the aqueous solution a desiredshape, and then removingall, or most of the water. Films are thusprepared by casting an aqueous pullulan solution on a flat, horizontalsubstrate, and evaporating the water. Other thin-walled shapes havingdimensions of length and width which are many times greater than thethickness of the body are produced in an analogous, essentiallyconventional manner. Pullulan is pseudothermoplastic and may thus becompression molded or extruded at elevated temperature.

Films and sheets of pullulan are transparent'and colorless, and freesurfaces formed by evaporation of water from a pullulan solution have ahigh gloss. The tensile strength of shaped bodies of pullulan is of thesame order of magnitude as that of regenerated cellulose (Cellophane)and may be higher at low moisture content. Films and sheets of pullulanare pliable and have high folding endurance. The mechanical and opticalproperties of the material are not impaired by aging and storage at veryhigh or very low relative humidity.

Although the material is soluble even in cold water, it

does not become tacky when exposed to a moist atmosphere. It does notbecome brittle at temperatures as low as lOC. It is not affected by oiland insoluble in many common organic solvents. lt does not retaincharges of static electricity and does not readily support the growth ofmicroorganisms.

Even very thin films of pullulan are almost impermeable to atmosphericoxygen. The material is relatively permeable to water vapor.

As is inherent in the above list of properties, pullulan films,coatings, sheets, and the like are valuable packaging materials,particularly for materials that need to be protected against atmosphericoxygen. If protection against atmospheric moisture is also required, itcan readily be provided by a secondary envelope of other availablepolymers such as polyethylene. Conforming coatings of pullulan areprepared by spraying the object to be protected with an aqueous solutionof pullulan an by thereafter evaporating or otherwise removing thewater. As will be shown hereinbelow, a frozen envelope prepared byquickly cooling an aqueous pullulan solution has properties closelysimilar to those of a sheet of substantially water-free pullulan.

Pullulan is compatible with other water-soluble, filmforming polymers,such as amylose, polyvinyl alcohol, and gelatin, and shaped bodiesconsisting of mixtures of pullulan with such other film-formingingredients are prepared in the same manner as the afore-describedpullulan bodies, that is, by compression molding of a particulate,intimate mixture of the ingredients, or by evaporation of water from acommon aqueous solution. The valuable properties of pullulan areretained to an important extent if the mixtures do not contain more than120 percent amylose, percent polyvinyl alcohol, and/or 150 percentgelatin, based on the weight of the pullulan in the mixture. The:effects of the additional film-forming ingredients on the mechanical,optical, and physical properties 'of pullulan will be illustratedhereinbelow by specific examples.

Bodies consisting essentially of pullulan or of mixtures of pullulanwith amylose, polyvinyl alcohol, and- /or gelatin may be plasticized bymeans of polyhydric alcohols. While any polyhydric alcohol is effectiveas a plasticizer for shaped bodies of pullulan or its aforedescribedmixtures, maltitoLsorbitol, and glycerol have been found most effective.Water-soluble grades of polyvinyl alcohol also act as plasticizers. Theamounts of plasticizer employed depend on the nature of the polymericingredients, on the result that it is desired to achieve, and on thenature of the plasticizer. Generally, no significant change inproperties is observed with less than 1 percent of plasticizer, based onthe weight of the shaped body, and some of the desirable properties ofpullulan are lost when the plasticizer concentration exceed's 20 percentof the body weight unless relatively large amounts of gelatin arepresent. 1

Films and coatings of pullulan are superior to amylose films in theirresistance to aging, in their optical properties, in theirimpermeability to oxygen, and in their ability of dissolving in waterquickly and without leaving a trace.Typically, a pullulan film up to athickness of 0.2 mm vanishes in contact with water at 30C within afraction of a minute, andthe dissolution rate increases with the watertemperature. It is not materially reducedby admixture of-thefilm-forming materials mentioned above in the amounts specified.

The degree of polymerization and molecular weight of pullulan vary withthe specific strain of Pullularia employed in preparing thepolysaccharide. Pullulan having a molecular weight of about 250,000 hasbeen found to produce shaped bodies of most desirable mechanicalstrength while producing aqueous solutions of relatively high viscosity.Some mechanical strength is lost in pullulans of lower molecular weight,such as 50,000, but more concentrated aqueous solutions are readilyprepared. The aqueous solutions employed in making films and otherpredominantly two-dimensional shapes of pullulanpreferably contain 3percent to 10 percent of the material, and they may additionally containamylose, polyvinyl alcohol, and/or gelatin.

Relatively concentrated solutions of pullulan are prepared mostconveniently from pullulan recovered from a culture medium withoutdrying. Such wet pullulan readily disperses in hot water. Thoroughlydried pullulan in pieces of substantial thickness may require atemperature as high as C or even more to dissolve within a short time.

Films may be cast from aqueous solutions containing 5 percent pullulanof molecular weight 250,000 on smooth glassor metal plates in aconventional manner, and the viscosity of the castingsolution may be adjusted by varying the concentration and/or temperature for producingfilms of desired dry thickness. The fluid coating initially cast may bedried by a stream of hot air whose temperature is not critical. Noturbidity is caused even at relatively high air temperature. The viscouspullulan solutions tend to entrap air in minute bubbles, and suchbubbles must be removed by applying a vacuum to the casting solution ifa very thin film free from pinholes and visible blemishes is to beproduced.

Obviously, other conventional methods of forming films from solutions offilm-forming ingredients in volatile solvents are applicable to pullulanand to its mixtures described above. Films having a thickness between0.01 and 0.2 mm are readily prepared by casting.

A pullulan film 0.1 mm thick typically transmits 95% of incidentsunlight, has a tensile strength of 7 8 kg/mm, an elongation of 8 to 20percent, and a folding endurance of 800 900 cycles of double folding.After one month storage at 60 percent relative humidity and 25C, thetensile strength is unaffected, the elongation may drop to 8 to l 1percent, and the folding endurance to 700 750 cycles at 30C, and to 550600 cycles at 10C. These values are obviously far superior to those ofamylose films, the only other films that are known to be water solubleat least to some extent, oil resistant, and edible.

The oxygen permeability of thin pullulan film is 1 2 mllm lday at 29 1Cas determined by A.S.T.M. Method D-l434.

The equilibrium moisture content of pullulan films varies relativelylittle with variations in ambient atmospheric moisture, and the materialdoes not become tacky at relative humidity values as high as 80 percent.

When polyvinyl alcohol is used in a mixture with pullulan as afilm-forming agent, it is preferred to choose polyvinyl alcohol having aviscosity of 10 27 cps in 4 percent aqueous solution at 20C, and asaponification value of 87 98 percent. A'rnylose, when mixed with thepullulan, is preferably free, or practically free of amylopectin.Gelatin increases the elastic modulus of pullulan films, and relativelylarge amounts of gelatin may cause some brittleness which can beovercome by the use of plasticizers, as much as 30 percent plasticizerbeing advisable and useful in films consisting of pullulan and equal orgreater amounts of gelatin. Under other conditions, an amount ofplasticizer greater than 20 percent is not conducive to best mechanicalproperties.

When the water solubility, stability over an extended period of time andover a wide range of temperatures, particularly low temperature, hightransparency, high gloss, and nonpermeability to oxygen which arecharacteristic of pure pullulan films are to be retained to theirfullest extent, the combined amount of amylose, polyvinyl alcohol, andgelatin in a film should not exceed 50 percent of the pullulan weight.

Because of their good flexibility at very low temperature and theirimpermeability to oxygen, the pullulan films and films of pullulanblends are eminently suitable for packaging frozen food. The oilresistance of the films makes them particularly useful for packagingbutter, cheese, and other fat-bearing foods. Pharmaceuticals and enzymeswhich deteriorate in contact with atmospheric oxygen may be stored forextended periods when enveloped in films of pullulan or pullulan blends.

Plates and other shaped bodies such as filaments and fibers can beproduced by utilizing the pseudo-thermoplasticity of pullulan. Purepullulan containing less than about 25% moisture can be compressionmolded or extruded at C at pressures of 100 kg/cm The temperature andpressure may have to be modified in the presence of other polymers, suchas amylose or polyvinyl alcohol, and in the presence of plasticizers.Pulverulent mixtures of pullulan with amylose and/or polyvinyl alcoholcontaining 10 percent 20 percent moisture and not more than 5 percentplasticizer have been molded and extruded successfully.

The fibers or filaments so obtained are of limited direct applicabilitybecause of their water solubility. They can be made water resistant in aknown manner by treatment with glyoxal or formaldehyde, or by theprovision of a waterproof coating.

Cotton dyes are generally absorbed by pullulan, and colored pullulanbodies may be produced from colored aqueous solutions or by moldingsubstantially dry mixtures containing pigments. Pullulan may be used toadvantage in textile sizing to reduce the build-up of static electricityon fibers, particularly synthetic fibers, during processing.

The following Examples are further illustrative of the invention, and itwill be appreciated that the invention is not limited thereto.

EXAMPLE 1 An aqueous culture medium containing, by weight, 10 percentsucrose, 0.5% K HPO 0.1% NaCl, 0.02% MgSO .7H O, 0.06% (NH,) SO and 0.04percent yeast extract was sterilized 20 minutes at 10 p.s.i. and thencooled. it was inoculated with a loopful of a culture of Dematiumpullulans IFO 4464 preveiously produced on a 1.5 percent agar medium ofotherwise the same composition in one week at 24C. The inoculated mediumwas incubated at 27C for one week with shak- The microbial cells werethen removed by centrifuging, and the supernatant liquid was mixed withan equal weight of methanol to precipitate the pullulan formed byfermentation. It was whitish and readily recovered by decantation,washed with methanol and dried. It had a mean molecular weight of250,000 and a specific rotation of [01],, It was identified by itsdecomposition to maltotriose by pullulanase.

This pullulan will be referred to hereinbelow as pullulan A.

EXAMPLE 2 An aqueous culture medium containing, by weight, 3 percentglucose, 0.12 percent urea, 0.1 percent yeast extract, 0.5% K HPO and0.08 percent MgSO .7l-l O was sterilized as described in Example 1,inoculated with a strain of Pullularia pullulans lFO 6353, and incubatedat 27C for one week with shaking, whereby it was converted to amucilaginous mass from which the cells were removed and pullulan wasprecipitated by means of methanol.

The product so obtained had a molecular weight of 60,000 and an opticalrotation of M1,, 171. It yielded maltotriose when decomposed bypullulanase and will be referred to hereinbelow as pullulan B. lts lowermolecular weight is believed due to amylase produced by the microbialstrain employed, and a longer culturing period would have producedpullulan of even lower degree of polymerization.

EXAMPLE 3 Pullulan A was dispersed in Water at 90C with stirring toproduce a 5 percent solution, all percentage values herein being byweight unless specifically stated otherwise. The solution was cooled to50C, deaerated by exposure to a vacuum, cast on a clean steel plate in auniform thickness, and dried in an air stream at 70C.

The film so obtained had a thickness of 0.02 mm, and was transparent,colorless, glossy, flexible, non-tacky, and tough. Small pieces placedin water at 30C swelled immediately, disintegrated, and disappeared bydissolu tion within about 20 seconds.

Its tensile strength was 7.1 kglmm the elongation percent, and itwithstood 700 cycles of folding under standardized conditions. Nomeasurable change in elongation and folding endurance was observed afterstorage for one month at 60 percent relative humidity. Tensile strengthimproved slightly to 7.2 kglmm EXAMPLE 4 Pullulan B was dispersed withstirring in hot water for minutes to produce a homogeneous 6 percentsolution. 2 percent Maltitol, based on the pullulan weight, was added tothe solution with stirring. The solution was deaerated, cast on a cleanmetal plate at 60C, and dried in an air stream at 80C to produce a softfilm 0.02 mm thick whose transparency and gloss were very good and onlyslightly inferior to the film prepared in Example 1, while the film wasslightly more readily soluble in water. Its tensile strength was 6.5kglmm the elongation 21 percent, and it withsood 780 of the foldingendurance test cycles. These properties were virtually unaffected by onemonth storage at 60 percent relative humidity.

An increase in the maltitol concentration to 5 percent decreased thestiffness and toughness of the film while greatly increasing theextensibility. The more plasticized material was found suitable formaking soft capsules for pharmaceutical use and for coatings. Closelysimilar produced were obtained when the ma]- titol was replaced by equalamounts of glycerol or sorbitol, but some surface smoothness was lost.

EXAMPLE 5 An aqueous 7 percent solution of pullulan A was prepared bystirring the ingredients at 100C. An aqueous 7 percent suspension ofgelatin was heated at 80C until uniform. Three parts of the pullulansolution and one part of the gelatin solution were mixed, and maltitoland sorbitol were added in amounts of l percent each, based on theweight of the film-forming ingredients, pullulan and gelatin. Thesolution was deaerated, cast on a metal plate at 70C, and dried in anair stream at 80C.

The film so produced had a thickness of 0.02 mm, a transparency of 92percent as compared to 95 percent for that of Example 3, and good gloss.It dissolved in water at 30C in 20 21 seconds, had a relatively hightensile strength of 7.5 lag/mm combined with an elongation of 13 percentand a folding endurance of 560 cycles. These properties were notsignificantly affected by storage for one month at 60 percent relativehumidity and 25C. The oxygen permeability of the film was slightlyinferior to the film of Example 3.

EXAMPLE 6 Amylomaize starch hydrolyzed with isoamylase percent amylosecontent) was heated in water to C for 10 minutes to produce a 5 percentsolution. Four parts of a 5 percent solution of pullulan A, prepared asin Example 3, and one part of the amylose solution were mixed whilewarm, and 1. percent maltitol based on the weight of the film-formingingredients was added. The solution was deaerated, and a film was formedby quickly drying a cast layer of the solution.

The dry film had a thickness of 0.02 mm. a transparency of 93 percent,and very good gloss. It dissolved in water at 30C in 20 23 seconds, hada tensile strength of 6.8 kg/mm an elongation of 15 percent, and afolding endurance of 680 cycles. Its mechanical properties wereunaffected by one month storage at 60 relative humidity.

EXAMPLE 7 Gelatinized starch was hydrolyzed and further exposed toisoamylase to produce an amylose of which 50 percent had a degree ofpolymerization below 50. Respective aqueous 8 percent solutions ofpullulan B and of the low D.P. amylose were mixed in a ratio of 10:1 atelevated temperature, and the practically clear mixture was deaeratedand cast on a metal plate.

The film produced by drying had a thickness of 0.02 mm, a transparencyof 93 percent, good gloss, and dissolved in water at 30C in theparticularly short time of 18 seconds. Its tensile strength of 6.5 kg/mmdecreased in one month of storage at 60 percent RH. and 25C to 6.1 kglmmbut elongation and folding endurance were unchanged at 1 1 percent and650 cycles respectively.

EXAMPLE 8 A 5 percent solution of pullulan A in hot water was mixed with15 percent amylose based on the weight of the pullulan. The amylose wasprepared by hydrolyzing a liquefied starch solution with iso-amylase,and precipitating the fraction containing more than 50 percent amyloseof D.P. 50 or higher. The mixed solution was heated and stirred untiluniform, and maltitol and sorbitol were added as plasticizers inrespective amounts of 1 percent of the film-forming ingredients. Thesolution was deaerated, cast on a metal plate, and dried as in thepreceding Examples.

The film, having a thickness of 0 .02 mm, had a transparency of 93percent, good gloss and solubility in water at 30C (20 22 seconds), atensile strength of 6.5 kglmm an elongation of 20 percent, and a foldingendurance of 720 cycles. Storage at 60 percent RH. for one month had noeffect on the mechanical properties.

EXAMPLE 9 Polyvinyl alcohol (PVA) having a viscosity of 20 cps and asaponification value of 88 was mixed with an aqueous 4 percent solutionof pullulan A in an amount of 30 percent based on the pullulan. Ahomogeneous solution was formed by heating and stirring. It wasdeaerated and cast on a metal plate and dried to form a film 0.02 mmthick.

The transparency of the film was 94%, its gloss high, and it dissolvedin water at 30C in 20 22 seconds. Its

tensile strength of 6.6 kg/mm and elongation of 16 percent were notsignificantly affected by storage for one month at 60 percent R.H., butfolding endurance increased from 670 to 750 cycles.

Because of its solubility, the intimate mixture of PVA and pullulan wasparticularly well suited for preparing a coating solution in which nutswere dipped and dried. The coated nuts retained their taste,consistency, and flavor for an extended period of time.

EXAMPLE 10 An aqueous 5 percent solution of pullulan B was mixed withpolyvinyl alcohol having a viscosity of 28 cps and a saponificationvalue of 89 in an amount of percent based on the weight of the pullulanB. After the PVA was completely dissolved by heating and stirring,maltitol was added in an amount of 1 percent based on the film-formingingredients, and the solution was deaerated and cast on a metal plate.

The film formed after drying had a transparenecy of 94 percent, goodgloss, and readily dissolved in warm water. Its tensile strength was 6.5kg/mm the elongation 14 percent, and the folding endurance 620 cycles.These properties were unaffected by storage at 60 percent R.H. for onemonth. The film had particularly low permeability to atmospheric oxygen.

EXAMPLE 1 l A solution of 5 percent pullulan A in water was mixed with20 percent polyvinyl alcohol, based on the pullulan weight, the PVAhaving a viscosity of l 1.8 cps and a saponification value of 98. Thefilm of 0.02 mm prepared from this solution had a transparency of 93percent, good gloss, and dissolved in water at 30C within 18 20 seconds.lts tensile strength was 6.0 kg/mm the elongation 13 percent, and thefolding endurance 720 cycles. No significant changes in the mechanicalproperties were observed after storage at 60 percent RH. for one month.

EXAMPLE 12 An aqueous 5 percent solution of pullulan B was mixed at 80Cwith 50 percent water soluble polyvinyl alcohol, based on the pullulanweight, the PVA having a viscosity of 27 cps and a saponification valueof 98. An adequately homogeneous solution was produced within threeminutes, deaerated, and converted into a film 0.02 mm thick as describedin the preceding Examples and having similar properties.

When 2 percent maltitol, based on the weight of the film-formingingredients, was incorporated in the solution prior to casting, a soft,transparent, glossy film was obtained.

EXAMPLE 13 An intimate and uniform mixture 'was prepared from fourparts, by weight, pullulan powder containing 15 percent moisture and onepart gelatin (6 percent moisture), also 0.5 percent maltitol based onthe combined weight of pullulan and gelatin. Sheets 3 mm thick wereprepared from the mixture at 120C on a laboratory press. They werewhite, semi-opaque, and oil resistant, but attackable by the water.

EXAMPLE l4 A pullulan C having a slightly lower molecular weight thanpullulan B and lower viscosity was prepared in a manner analogous toExample 2, and could be dissolved in water to form a fluid 10 percentsolution. A 10 percent solution of amylose in water was preparedseparately from a starch containing percent amylose whose 10 percentsolution was gelatinized at 130C and thereafter hydrolyzed at pH 4 bymeans of isoamylase derived from Pseudomonas. When the hydrolyzationmixture was cooled, amylose was precipitated and was employed inpreparing the hot, aqueous, 10 percent solution of which one part wasmixed with three parts of the pullulan C solution. 2 percent Sorbitol,based on the weight of the film-forming ingredients, was added.

The solution so obtained could be sprayed from an air gun for coatingfresh food, pharmaceuticals, enzyme particles, dehydrated food and likeoxidizable products with a film practically impervious to atmosphericoxygen and sufficiently strong to prevent mechanical damage to thecoated articles. Water-soluble capsules for pharmaceutical purposes wereformed from the same solution by dipping metal rods having sphericallyrounded ends in the solution and drying the liquid film formed on therods withdrawn from the solution in an air stream at 40C.

EXAMPLE l5 Amylose prepared as described in Example 9 was suspended inan aqueous gelatin solution at C in an amount equal to the weight of thegelatin, and enough wet pullulan B was added to the suspension to makethe weight ratio of pullulan, gelatin, and amylose 6:212 on a dry basis.The mixture was stirred at C until a homogeneous solution was obtained.Films were prepared by casting the solution on metal plates and removingmost of the water present by a stream of air at 50C.

The films stripped from the metal plates were transparent, smooth, andglossy, and readily dissolved in water. When in moisture equilibriumwith ambient air, they were tough and resilient.

EXAMPLE 16 Three parts of an aqueous 7 percent solution of pullulan Aand one part of a 20 percent gelatin solution were mixed at 70C. Themixture, which contained approximately equal weights of pullulan andgelatin, was deaerated, and capsules were prepared by the methoddescribed in Example 14.

Capsules were also prepared from a solution of equal parts of pullulanand gelatin which additionally contained 1 percent maltitol, based onthe film-forming ingredients. Both batches of capsules had therelatively high modulus of elasticity characteristic of pharmaceuticalcapsules commonly referred to as hard" capsules. They were strong enoughnot be deformed when stacked in a pile. The plasticized capsules showedeven greater breaking strength than those prepared from pullulan andgelatin alone.

EXAMPLE 17 Ten g Aliquots of sardine oil, oleic acid, and linoleic acidwere adsorbed on equal amounts of diatomaceous earth purified bytreatment with aqua regia, and the samples so obtained were packed inidentical bags of pullulan film 0.05 mm thick. The bags were sealed in avacuum and stored at 35C. The contents of sample bags were extractedwith chloroform immediately after sealing, after 3 days of storage, andafter 14 days of storage.

The deterioration of the samples by oxidation was determined fromoptical density readings taken under 10 polyethylene film and a commonpolyethylene bag and stored in the same incubator.

The activity of theyeast before and after storage was determined by theamount of carbon dioxide developed uniform conditions at 530 mp. and450mp. TBA (2- from 3 percent sucrose solution. The yeast stored inthiobarbituric acid)-value initially, after 3 days, and pullulanretained92 percent of its initial activity, that after 14 days, bydetermining the peroxide value inistored 1n polyethylene showed only 40percent of its intially andafter 3 clays and by measuring opticaldensity ma] actlvlty after Storageafter 14 days at 420 mu, a wavelengthcharacteristic of Samples f detergent contammg alkalme Proteaseyellowing. The results are tabulated below together weltefested an P pfi with those obtained on controls whichwere either sup actwlty wasdetermmed the onglrgal fnaterlal and ported or containedin bags ofregenerated cellulose the sample Stored one monlh f C pullulan and(Cellophane) and polyethylene 0'05 mm thick; polyethylene envelopesrespectively. The pullulan en- Optical Density at 530 mp. OpticalDensityat 450 mp.

0|.ac. Lin.ac. Sard.0. OLac. Lin.ac. Sard.O.

Initial 0.015 0.032 0.034 0.002 0.007 0.002

After 3 days in pullulan 0.052 0.067 0.073 0.023 0.014 0.038 in Celloph.0.065 0.105 0.180 0.025 0.018 0.21 in polyeth. 0.07 0.13 0.23 0.023 0.250.31 unprotected 0.07 0.2 1.925 0.023 0.985 0.54

after 14 days in pullulan 0.13 0.13 1.28 0.03 0.04 0.057 in Celloph.0.91 2.30 2.50 0.33 0.75 0.15 in polyeth. 1.25 1.15 2.51 0.31 0.21 1.20unprotected 2.88 2.20 4. 55 0.97 0.62 1.24

optical density ofTBA-valuc per 0.1 of fat.

Table 2 velopes prevented'sig nificant loss of protease activity whereasthe samples stored in polyethylene were re- Peroxide valu meq/kg ducedto 60 percent of theiroriginal enzymatic activity. Oleic Acid LinoleicA. Sardine Oil 35 One gram samples of ascorbic acidand of riboflavinlmtlal 0.74 2.47 0.76

After3 days were vacuum-sealed 1n pullulane film envelopes, l g' ,2: 1-mm X mm, which were further protected by a polym op ane ethylene bag,andcontrols-were enclosed in polyethylpolyethylene ene envelopes ofequal 'size andjan. outer polyethylene Pppmecled 199-5 bag. After fivemonths of storage at 35C, the samples of Vitamin C still had94:percentand those of Vitamin "3 B 96 percent of their original activity whenstored in pullulan, while the activity of the samples stored inyellowing polyethylene had dropped to 31 percent and 45 per- Optical l)ensity at 420 m,r after 14 Days cent respectively. Oxidation sensitivemechanical com- Sa'dme pounds are conveniently protected by placingindividl bio 0.22 ual doses ona first pullulansheet 1n rows andcolumns,

in Cellophane 0,1 0, 015 covering the first sheet and the mater1althereon by a g P Y y1i 8P g-5 5 second sheet of pullulan,heat-sealingthe two sheets to pmtecte eachother in a rectangular grid ofseams between the rows and columns, and then severing the sealed sheetsThe results numerically expressed in Table 3 confirm. through the seams,therebyproduc1ng1nd1v1dual sealed the results of visual inspection.Oleic acid appeared 9. .9. 3 Th Bll l at ill may b mgested togetherwhite when stored in pullulan or Cellophane bags, w1th their contentssmce'they are water-soluble and weakly yellow when stored inpolyethylene or without 4 protect1on. Sardme o1lwas weakly yellow afterstorage EXAMPLE 1n pullulan bags, more strongly yellow when stored in I1 Cellophane bags, and deeply yellow o brow i h h Powdered soup mix wasvacuum packed m 1nd1v1dual stored in polyethylene or unprotected;portion amounts in envelopes of pullulan film 0.05 mm I thick, and adozen envelopes were further enclosed in EXAMPLE 8 a polyethylene bagfor protection against moisture and Five g Samples of bakery yeast werepacked inenvelmicrobial infestation. Each envelope, when dropped opes,30 mmX 50 mm, of pullulan film0.05 mm thick, into approximately 8 ozs.of hot water, produced a porand the envelopes were heat-sealed in avacuum. 20 tion of soup to which the pullulan film contributed nei-Sealed envelopes were sealed in a polyethylene bag for moistureprotectionandstored in an incubator for one month at 35C. Controls weresealed in envelopes of ther taste nor odor nor any other perceivableproperty. After six months of storage under ambient conditions,

the pullulan-packed soup wasdistinctly superior in its.

wet coating. A second batch was e flavor and texture to conventionallypackaged batches of the same soup mix.

The same beneficial effects of a pullulan envelope were observed withinstant Chinese noodles. The mixed ingredients, including Shiitake, anedible mushroom, vegatables, and shrimps were placed in a flat layer ona pullulan film and then sprayed with a pullulan solution from which thewater was removed thereafter by an air stream at 50C. The laminar sheetso produced was cut into pieces of convenient size which were enclosedin a poyethylene bag, and were ready for cooking when withdrawn from thebag.

EXAMPLE 20 ,change in color, increased butyric acid odor, and asignificant deterioration in their taste. The perioxide value of thewrapped butter samples was 20.3 meq/kg, that of the unprotected samples46.5 meq/kg. The unprotected cheese samples showed a small, but clearlydiscernible deterioration of their taste, odor, and color, and they weredried out. I

EXAMPLE 21 Freshly baked doughnuts were sprayed with a pullulansolution, and the coating formed thereby was dried at 60C for minutes toa thickness varying between 0.05,and 0.1 mm. Several coated doughnuts,uncoated doughnuts in pullulane bags, and uncoated controls were packedin separate polyethylene bags and stored at 35C for one week. Thedoughnuts protected by a sprayed coating or a separate bag of pullulanwere still in saleable condition, retaining their initial moisture andtaste, whereas the controls were distinctly stale.

Pullulan is particularly effective in preventing rancidity of fat inbaked or fried goods. Fried crackers and peanuts roasted in butter stillhad their fresh smell and unchanged taste after 40 days storage underambient conditions in envelopes of pullulan film 0.03 mm thick, furtherprotected by polyethylene bags, whereas rancidity was obvious incontrols packed in two layers of polyethylene.

Fresh mackerel was sprayed with a 4 percent pullulan solution containing3 percent glycerol based on the weight of the pullulan, and the sprayedfish was transferred immediately to a freezer kept at C. Samples ereksntftq the oa ed fish n m an unspa ed control after three monthsstorage in which the pullulan solution had formed a frozen, shinyenvelope on the fish. The envelope was strong enough to resist manualremoval, but disappeared on contact with hot water. The taste of thecooked, pullulan-protected fish was distinctly superior to that of thecontrol, and the peroxide value of the skin of the coated mackerel wassignificantly lower than the corresponding value determined on thecontrol.

' EXAMPLE 22 Freshly baked biscuits were coated with a thin film ofpullulan by spraying a 3 percent solution and drying the nclosed inpullulan bags, and a third batch of the same biscuits was used as acontrol. Each batch was packed in a polyethylene bag and stored twomonths at 30C in a chamber kept at 60 percent relative humidity.

The biscuits protected by pullulan retained their fresh taste whereassome rancidity was noticeable in the controls. The coated samples,moreover, retained their shape during storage and handling while theuncoated biscuits showed evidence of crumbling. The clear pullulan filmgave gloss to the coated samples, but was not felt during eating of thebiscuits.

EXAMPLE 23 Hams and sausages were steam-heated in watersoluble casingsand molded, whereupon the casings were removed. The batch was dividedinto two portions of which one was uniformly sprayed with warm 5 percentpullulan solution to produce a film which had a thickness of about 0.05mm after drying in an air stream at C. The coated harms and sausagestogether with the unprotected controls were stored at 5C for threemonths which did not cause cracks or turbidity in the pullulan films onthe coated pieces.

The aldehyde content of the coated samples and the controls was measuredby the TBA method, and only one-third to one-fifth of the aldehydecontent of the controls was found in the coated material which isindicative of the oxidation protection afforded by the thin pullulanfilm.

EXAMPLE 24 Freshly harvested spinach was sliced and freeze-dried at-20C. Samples of the product were placed on a metal plate in a vacuumdrier at 30C and mm Hg and sprayed with a pullulan solution so that thedried spinach and the plate were enveloped by a film of pullulan. Thecoated spinach was further protected by being sealed in a polyethylenebag. When reconstituted after five months, the sinach wasindistinguishable in color, shape, odor, and flavor from spinachreconstituted immediately after freeze-drying.

Alternatively, fresh spinachwas cut into slices and sprayed with a 3percent pullulan solution. It was then freeze-dried while still wet, andsealed in a polyethylene bag. The appearance, taste, and odor of thespinach when reconstituted five months later was not different from thatof spinach reconstituted immediately after freeze-drying. The vitaminloss in storage was only 10 percent.

EXAMPLE 25 Beef sliced to a thickness of 5 to 10 mm was seasoned, androasted. The roasted slices were sprayed with hot 7 percent pullulansolution to a thickness sufficient to produce a film without pinholes,and the coated slices were immediately freeze-dried. Control slices werefreeze-dried as roasted. Samples of the coated slices and the controlswere stored for 50 days in each of two incubators respectively kept at35 and 65C.

Samples were withdrawn at 10 day intervals and analyzed for oxidation ofthe fat content by determination of acid value, peroxide value, andaldehyde content, the latter being determined by measuring the opticaldensity according to the TBA method. Extracts of the fat were preparedby grinding a 10 g sample in a mortar and triturating the ground samplewith 200 g of a 2:1

(volume) mixture of chloroform and methanol. The extract was dried andevaporated at 35C in a vacuum. The moisture content of the freeze-dried,coated samples was 4 7 percent, that of the uncoated controls 2 4percent. The results of the tests on products stored at 35 and 65Crespectively are listed in Tables 4 and 5.

Table 4 (35C) Sampled after I0 Acid value in coated slices uncoatedPeroxide value in coated slices uncoated Aldehyde in (TBA'value) coatedslices uncoated 0.l8

Table s (65C) The numerical values listed in Tables 4 and 5 wereconfirmed by subjective tests. Samples were heated in an electronic oventogether with 10 percent water and permitted to stand for ten minutes.They were then tasted. The pullulan coatings were dissolved in the hotwater, and the samples protected by a pullulan film during storage werefound to be consistently superior to the uncoated slices in flavor andtaste.

While the invention has been described by reference to specificembodiments, it should be understood that it is not limited to thespecific examples chosen for the purpose of the disclosure, but is to beconstrued broadly and limited solely by the scope and spirit of theappended claims.

What is claimed is:

l. A shaped solid body consisting essentially of a uniform mixture ofpullulan with at least one member of the group consisting of amylose,polyvinyl alcohol, and gelatin, the amount of said amylose being notgreater than 120 percent of theweight of said pullulan, the amount ofsaid polyvinyl alcohol being not greater than 100 percent of the weightof said pullulan, and the amount of said gelatin being not greater than150 percent of the weight of said pullulan, said pullulan having amolecular weight of approximately 50,000 to approximately 250,000, andbeing a polysaccharide essentially consisting of maltotriose unitslinked by 01-1 ,6-bonds.

2. A body as set forth in claim 1, further containing an amount of apolyhydric alcohol sufficient to plasticize said mixture.

3. A body as set forth in claim 2, wherein said polyhydric alcohol isglycerol, sorbitol, or maltitol.

4. A body as set forth in claim 3, wherein said polyhydric alcoholconstitutes 1 percent to 20 percent of the weight of said body.

5. A body as set forth in claim 1 having dimensions of length and widthmany times greater than the thickness thereof.

6. A method of making the body set forth in claim 1 which comprisesdissolving said pullulan and said at least one member in water,imparting a shape to the aqueous solution so produced, and substantiallyremoving the water from said solution.

7. A method of making the body set forth in claim 1 which comprisesshaping said mixture above the softening temperature thereof underapplied pressure.

8. A body as set forth in claim 1,, wherein said pullulan is a productof fermentation by means of Pullularia pullulans or Dematium pullulans.

9. A shaped solid body consisting essentially of pullulan and an amountof a polyhydric alcohol sufficient to plasticize said pullulan, saidpullulan being a polysaccharide essentially consisting of maltotrioseunits linked by a-l,6-b0nds'and having a molecular weight ofapproximately 50,000 to approximately 250,000.

10. A body as set forth in claim 9, which has a transparency of morethan percent in a thickness of 0.1 mm, a tensile strength notsubstantially smaller than 6.0 kg/mm and an elongation of at least 8percent after storage for one month at 60 percent relative humidity and25C.

11. A body as set forth in claim 9., wherein said pullulan is a productof fermentation by means of Pullula'ria pullulans or Dematium pullulans.

12. A body as set forth in claim 9, wherein said polyhydric alcohol isglycerol, sorbitol, or maltitol in an amount constituting 1 percent to20 percent of the weight of said body.

13. A method of making the body set forth in claim 9, which comprisesdissolving said pullulan and said alcohol in water, imparting a shape tothe aqueous solution so produced, and substantially removing the waterfrom the shaped solution.

14. A method of making a shaped body essentially consisting of pullulanwhich comprises shaping said pullulan above the softening temperaturethereof under applied pressure, said pullulan having a molecular weightof approximately 50,000 to approximately 250,000 and being apolysaccharide essentially consisting of maltotriose units linked bya-l,6 bonds.

2. A body as set forth in claim 1, further containing an amount of apolyhydric alcohol sufficient to plasticize said mixture.
 3. A body asset forth in claim 2, wherein said polyhydric alcohol is glycerol,sorbitol, or maltitol.
 4. A body as set forth in claim 3, wherein saidpolyhydric alcohol constitutes 1 percent to 20 percent of the weight ofsaid body.
 5. A body as set forth in claim 1 having dimensions of lengthand width many times greater than the thickness thereof.
 6. A method ofmaking the body set forth in claim 1 which comprises dissolving saidpullulan and said at least one member in water, imparting a shape to theaqueous solution so produced, and substantially removing the water fromsaid solution.
 7. A method of making the body set forth in claim 1 whichcomprises shaping said mixture above the softening temperature thereofunder applied pressure.
 8. A body as set forth in claim 1, wherein saidpullulan is a product of fermentation by means of Pullularia pullulansor Dematium pullulans.
 9. A shaped solid body consisting essentially ofpullulan and an amount of a polyhydric alcohol sufficient to plasticizesaid pullulan, said pullulan being a polysaccharide essentiallyconsisting of maltotriose units linked by Alpha -1,6-bonds and having amolecular weight of approximately 50,000 to approximately 250,000.
 10. Abody as set forth in claim 9, which has a transparency of more than 90percent in a thickness of 0.1 mm, a tensile strength not substantiallysmaller than 6.0 kg/mm2, and an elongation of at least 8 percent afterstorage for one month at 60 percent relative humidity and 25*C.
 11. Abody as set forth in claim 9, wherein said pullulan is a product offermentation by means of Pullularia pullulans or Dematium pullulans. 12.A body as set forth in claim 9, wherein said polyhydric alcohol isglycerol, sorbitol, or maltitol in an amount constituting 1 percent to20 percent of the weight of said body.
 13. A method of making the bodyset forth in claim 9, which comprises dissolving said pullulan and saidalcohol in water, imparting a shape to the aqueous solution so produced,and substantially removing the water from the shaped solution.
 14. Amethod of making a shaped body essentially consisting of pullulan whichcomprises shaping said pullulan above the softening temperature thereofunder applied pressure, said pullulan having a molecular weight ofapproximately 50,000 to approximately 250,000 and being a polysaccharideessentially consisting of maltotriose units linked by Alpha -1,6 bonds.